1. Field of the Invention
This invention pertains to the field of compositions and methods that enhance the delivery of drugs and other compounds across the dermal and epithelial membranes, including, for example, skin, the gastrointestinal epithelium and the bronchial epithelium.
2. Background
Transdermal or transmucosal drug delivery is an attractive route of drug delivery for several reasons. Gastrointestinal drug degradation and the hepatic first-pass effect are avoided. In addition, transdermal and transmucosal drug delivery is well-suited to controlled, sustained delivery (see, e.g., Elias, In Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery, Bronaugh and Maibach, Eds., pp 1-12, Marcel Dekker, New York, 1989.). For many applications, traditional methods of administering drugs are not optimal because of the very large initial concentration of the drug. Transdermal delivery could allow a more uniform, slower rate of delivery of a drug. Moreover, patient compliance is encouraged because such delivery methods are easy to use, comfortable, convenient and non-invasive.
These advantages of transdernal and transmucosal delivery have not led to many clinical applications because of the low permeability of epithelial membranes, the skin in particular, to drugs. The difficulties in delivering drugs across the skin result from the barrier property of skin. Skin is a structurally complex thick membrane that represents the body""s border to the external hostile environment. The skin is composed of the epidermis, the dermis, the hypodermis, and the adenexal structures (epidermal appendages). The epidermis, the outermost epithelial tissue of the skin, is itself composed of several layersxe2x80x94the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale.
Compounds that move from the environment into and through intact skin must first penetrate the stratum corneum, the outermost layer of skin, which is compact and highly keratinized. The stratum corneum is composed of several layers of keratin-filled skin cells that are tightly bound together by a xe2x80x9cgluexe2x80x9d composed of cholesterol and fatty acids. The thickness of the stratum corneum varies depending upon body location. It is the presence of stratum corneum that results in the impermeability of the skin to pharmaceutical agents. The stratum corneum is formed naturally by cells migrating from the basal layer toward the skin surface where they are eventually sloughed off. As the cells progress toward the surface, they become progressively more dehydrated and keratinized. The penetration across the stratum corneum layer is generally the rate-limiting step of drug permeation across skin. See, e.g., Flynn, G. L., In Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery, supra. at pages 27-53.
After penetration through the stratum corneum layer, systemically acting drug molecules then must pass into and through the epidermis, the dermis, and finally through the capillary walls of the bloodstream. The epidermis, which lies under the stratum corneum, is composed of three layers. The outermost of these layers is the stratum granulosum, which lies adjacent to the stratum corneum, is composed of cells that are differentiated from basal cells and keratinocytes, which make up the underlying layers having acquired additional keratin and a more flattened shape. The cells of this layer of the epidermis, which contain granules that are composed largely of the protein filaggrin. This protein is believed to bind to the keratin filaments to form the keratin complex. The cells also synthesize lipids that function as a xe2x80x9ccementxe2x80x9d to hold the cells together. The epidermis, in particular the stratum granulosum, contains enzymes such as aminopeptidases.
The next-outermost layer of the epidermis is the stratum spinosum, the principal cells of which are keratinocytes, which are derived from basal cells that comprise the basal cell layer. Langerhans cells, which are also found in the stratum spinosum, are antigen-presenting cells and thus are involved in the mounting of an immune response against antigens that pass into the skin. The cells of this layer are generally involved in contact sensitivity dermatitis.
The innermost epidermal layer is the stratum basale, or basal cell layer, which consists of one cell layer of cuboidal cells that are attached by hemi-desmosomes to a thin basement membrane which separates the basal cell layer from the underlying dermis. The cells of the basal layer are relatively undifferentiated, proliferating cells that serve as a progenitor of the outer layers of the epidermis. The basal cell layer includes, in addition to the basal cells, melanocytes.
The dermis is found under the epidermis, which is separated from the dermis by a basement membrane that consists of interlocking rete ridges and dermal papillae. The dermis itself is composed of two layers, the papillary dermis and the reticular dermis. The dermis consists of fibroblasts, histiocytes, endothelial cells, perivascular macrophages and dendritic cells, mast cells, smooth muscle cells, and cells of peripheral nerves and their end-organ receptors. The dermis also includes fibrous materials such as collagen and reticulin, as well as a ground substance (principally glycosaminoglycans, including hyaluronic acid, chondroitin sulfate, and dermatan sulfate).
Several methods have been proposed to enhance transdermal transport of drugs. For example, chemical enhancers (Bumette, R. R. In Developmental Issues and Research Initiatives; Hadgraft J., Ed., Marcel Dekker: 1989; pp. 247-288), iontophoresis, and others have been used. However, in spite of the more than thirty years of research that has gone into delivery of drugs across the skin in particular, fewer than a dozen drugs are now available for transdermal administration in, for example, skin patches.
Transport of drugs and other molecules across the blood-brain barrier is also problematic. The brain capillaries that make up the blood-brain barrier are composed of endothelial cells that form tight junctions between themselves (Goldstein et al., Scientific American 255:74-83 (1986); Pardridge, W. M., Endocrin. Rev. 7: 314-330 (1986)). The endothelial cells and the tight intercellular junctions that join the cells form a barrier against the passive movement of many molecules from the blood to the brain. The endothelial cells of the blood-brain barrier have few pinocytotic vesicles, which in other tissues can allow somewhat unselective transport across the capillary wall. Nor is the blood-brain barrier interrupted by continuous gaps or channels that run through the cells, thus allowing for unrestrained passage of drugs and other molecules.
Thus, a need exists for improved reagents and methods for enhancing delivery of compounds, including drugs, across epithelial tissues and endothelial tissues such as the skin and the blood-brain barrier. The present invention fulfills this and other needs.
The present invention provides methods for enhancing delivery of a compound into and across one or more layers of an animal epithelial or endothelial tissue. The methods involve contacting tissue with a conjugate that includes the compound and a delivery-enhancing transporter. The delivery-enhancing transporters, which are also provided by the invention, have sufficient guanidino or amidino moieties to increase delivery of the conjugate into and across one or more intact epithelial or endothelial tissue layers compared to delivery of the compound in the absence of the delivery-enhancing transporter. Typically, the delivery-enhancing transporters have from 6 to 25 guanidino or amidino moieties, and more preferably between 7 and 15 guanidino moieties.
The delivery-enhancing transporters and methods of the invention are useful for delivering drugs, diagnostic agents, and other compounds of interest across epithelial tissues such as the skin and mucous membranes. Delivery across the blood-brain barrier is also enhanced by the conjugates and methods of the invention. The methods and compositions of the invention can be used not only to deliver the compounds to the particular site of administration, but also provide systemic delivery.
In some embodiments, the delivery-enhancing transporter comprises 7-15 arginine residues (SEQ ID NO:49) or analogs of arginine. The delivery-enhancing transporter can have at least one arginine that is a D-arginine and in some embodiments, all arginines are D-arginine. The delivery enhancing transporter can consist essentially of 5 to 50 amino acids, at least 50 percent of which are arginine. In some embodiments, at least 70% of the amino acids are arginines or arginine analogs. In some embodiments, the delivery-enhancing transporter comprises at least 5 contiguous arginines (SEQ ID NO:1) or arginine analogs.
The compound to be delivered can be connected to the delivery enhancing transporter by a linker. In some embodiments, the linker is a releasable linker which releases the compound, in biologically active form, from the delivery-enhancing transporter after the compound has passed into and through one or more layers of the epithelial and/or endothelial tissue. In some embodiments, the compound is released from the linker by solvent-mediated cleavage. The conjugate is, in some embodiments, substantially stable at acidic pH but the compound is substantially released from the delivery-enhancing transporter at physiological pH. In some embodiments, the half-life of the conjugate is between 5 minutes and 24 hours upon contact with the skin or other epithelial or endothelial tissue. For example, the half-life can be between 30 minutes and 2 hours upon contact with the skin or other epithelial or endothelial tissue.
Examples of conjugate structures of the invention include those having structures such as 3, 4, or 5, as follows: 
where R1xe2x80x94X comprises the compound; X is a functional group on the compound to which the linker is attached; Y is N or C; R2 is hydrogen, alkyl, aryl, acyl, or allyl; R3 comprises the delivery-enhancing transporter; R4 is substituted or unsubstituted S, O, N or C; R5 is OH, SH or NHR6; R6 is hydrogen, alkyl, aryl, acyl or allyl; k and m are each independently selected from 1 and 2; and n is 1 to 10. Preferably, X is selected from the group consisting of N, O, S, and CR7R8, wherein R7 and R8 are each independently selected from the group consisting of H and alkyl. In some embodiments, R4 is S; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, R2 is benzyl; k, m, and n are each 1, and X is O. In some embodiments, the conjugate comprises structure 3 and R2 is selected to obtain a conjugate half-life of between 5 minutes and 24 hours. In some embodiments, R2 is selected to obtain a conjugate half-life of between 5 minutes and 24 hours. In some embodiments, the conjugate comprises structure 4; R4 is S; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, the conjugate comprises structure 4; R5 is NHR6; R6 is hydrogen, methyl, allyl, butyl or phenyl; and k and m are each 1. One example of a conjugate is: 
where Ph is phenyl.
The invention also provides conjugates in which the release of the linker from the biological agent involves a first, rate-limiting intramolecular reaction, followed by a faster intramolecular reaction that results in release of the linker. The rate-limiting reaction can, by appropriate choice of substituents of the linker, be made to be stable at a pH that is higher or lower than physiological pH. However, once the conjugate has passed into and across one or more layers of an epithelial or endothelial tissue, the linker will be cleaved from the agent. An example of a compound that has this type of linker is structure 6, as follows: 
wherein R1xe2x80x94X comprises the compound to be delivered across one or more layers of an epithelial and/or endothelial tissue; X is a functional group on the compound to which the linker is attached; Ar is an aryl group having the attached radicals arranged in an ortho or para configuration, which aryl group can be substituted or unsubstituted; R3 comprises the delivery-enhancing transporter; R4 is substituted or unsubstituted S, O, N or C; R5 is OH, SH or NHR6; R6 is hydrogen, alkyl, aryl, acyl or allyl; and k and m are each independently selected from 1 and 2. In some embodiments, X is selected from the group consisting of N, O, S, and CR7R8, wherein R7 and R8 are each independently selected from the group consisting of H and alkyl. In some embodiments, R4 is S; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. In some embodiments, the conjugate comprises: 
In preferred embodiments, the compositions of the invention comprise a linker susceptible to solvent-mediated cleavage. For example, a preferred linker is substantially stable at acidic pH but is substantially cleaved at physiological pH.
Additional embodiments of the invention provide transdermal drug formulations. These formulations include a therapeutically effective amount of a therapeutic agent, a delivery-enhancing transporter that includes sufficient guanidino or amidino sidechain moieties to increase delivery of the conjugate across one or more layers of an animal epithelial tissue compared to the trans-epithelial tissue delivery of the biologically active agent in non-conjugated form; and a vehicle suited to transdermal drug administration.